Vaccine composition containing polyribosylribitol phosphate and method for making same

ABSTRACT

Vaccine compositions are disclosed which comprise at least one antigen formed by the capsular polysaccharide of Haemophilus influenzae type b or high molecular weight polyribosylribitol phosphate coupled to tetanus anatoxin.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of vaccine compositions and moreparticularly to vaccine compositions comprising at least one antigenformed by the capsular polysaccharide of Haemophilus influenzae type bor high molecular weight polyribosylribitol phosphate coupled to tetanusanatoxin.

2. Description of the Related Art

An antigen which can be used for vaccine purposes in man in order toprotect him from infections caused by Haemophilus influenzae type b isknown in the prior art, and especially through the article “Quantitativeand Qualitative Analyses of Serum Antibodies Elicited in Adults byHaemophilus influenzae Type b and Pneumococcus Type 6A CapsularPolysaccharide Tetanus Toxoid Conjugates” Rachel Schneerson et al,Infect. Immun. May 1986. This antigen is formed by a capsularpolysaccharide of the bacteria, polyribosylribitol phosphate (or PRP),which is made T-dependent owing to coupling to a carrier protein,tetanus anatoxin. Trials carried out on rhesus children have shown, asthis article reports, that the immune response was at one and the sametime greater and earlier if the antigen was associated with aluminiumhydroxide. However, as another article entitled “Clinical andImmunologic responses to the capsular polysaccharide of Haemophilusinfluenzae type b alone or conjugated to tetanus toxoid in 18-23month-old children”, Bo A. Claesson and al, The Journal of Pediatrics,May 1988, points out, it was noticed that this antigen, adsorbed onaluminium hydroxide, was less immunogenic after storage than the antigenkept in saline solution, which may be due to degradation of thepolysaccharide.

In order to resolve this problem of stability of the PRP-T, it wasproposed in the prior art to lyophilize it. This solution, even thoughit allows the antigen to retain its immunogenic character in the courseof time, shows some disadvantages, however, especially at themanufacturing level; the lyophilization and the particular operations ofconditioning which it requires complicates the production process, whichincreases the cost. In addition, at the time of administration, it isnecessary to take up the lyophilizate again, which means that it isnecessary to have in addition to the lyophilizate a liquid for taking upthis lyophilizate; this operation represents a supplementary constraintfor the practitioner and presents, like any manipulation, the risk ofbeing carried out badly.

In addition, a certain number of liquid vaccine combinations possessantigens adsorbed on an aluminium-based adjuvant and it would beadvantageous to be able, without loss of immunogenicity, to add theantigen formed by the PRP-T to them. In fact, the solution proposed inthe prior art and consisting in a special syringe with two compartments(a first compartment containing the PRP-T in lyophilized form and asecond compartment containing the other antigens in aqueous suspension)whose contents are mixed for use only at the time of administration isnot satisfactory either at the level of the costs of production or atthe level of the operations to be carried out by the practitioner.

It is thus desirable to be able to have a liquid vaccine compositioncomprising the antigen formed by the PRP-T having a very goodimmunogenic character retained in the course of time, and whoseconditions of manufacture allow production at the lowest cost.

SUMMARY OF THE INVENTION

This invention relates to vaccine compositions capsular polysaccharideof Haemophilus influenzae type b or high molecular weightpolyribosylribitol phosphate (PRP) coupled to tetanus anatoxin, as wellas an aluminium-based adjuvant. Such a combination permits a stable,liquid vaccine composition wherein each antigen retains itsimmunogenicity.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a vaccine composition comprising at least oneantigen formed by the capsular polysaccharide of Haemophilus influenzaetype b or high molecular weight polyribosylribitol phosphate coupled totetanus anatoxin as well as an aluminium-based adjuvant, characterizedin that the aluminium-based adjuvant has a point of zero charge of lessthan approximately 7.2.

It has thus been noticed that, surprisingly, under these conditions,PRP-T retains in the course of time in liquid medium its very goodimmunogenic character.

According to a particular characteristic of the invention thealuminium-based adjuvant comprises aluminium hydroxides to which anionshave been added.

Thus, it is possible to use an adjuvant perfectly qualified for vaccineuse while retaining in the PRP-T in liquid medium a very goodimmunogenicity.

According to another characteristic of the invention, the anions arechosen from amongst the phosphates or the citrates.

Thus, the composition obtained has all the safety guarantees necessaryfor a vaccine administration.

According to a particular mode of carrying out the invention, thevaccine composition additionally comprises one or more of the vaccinevalencies chosen from amongst: diphtheria, tetanus, whooping cough,hepatitis B and poliomyelitis.

It is thus possible to have liquid, stable vaccine combinations in whicheach antigen retains its immunogenicity, which allows the practitioner,without supplementary manipulation on his part, to vaccinatesimultaneously against several illnesses; this allows the costs to bereduced at one and the same time as far as the products are concernedand as far as the number of visits to be made are concerned.

The invention likewise relates to a method of manufacture of a vaccinecomposition comprising at least one antigen formed by the capsularpolysaccharide of Haemophilus influenzae type b or high molecular weightpolyribosylribitol phosphate coupled to tetanus anatoxin, characterizedin that it consists in adding an adjuvant to the vaccine composition bymeans of a suspension of aluminium complexes having a point of zerocharge of less than approximately 7.2.

The present invention will be better understood by reading the detaileddescription which will follow.

The antigen formed by the capsular polysaccharide Haemophilus influenzaetype b is a linear polymer consisting of ribose, ribitol and phosphoricacid which has the following monomeric structure:

The number of monomers of this type is high (greater than 100), whichleads to a polysaccharide whose molecular weight is of the order of500,000 to 1,000,000.

In order to induce an immune response of the T cells in young children,this antigen is conjugated to a carrier protein formed by tetanusanatoxin.

Such an antigen can, for example, be obtained according to the methoddescribed in “Quantitative and Qualitative analysis of serum antibodieselicited in adults by Haemophilus influenzae type b and Pneumococcustype 6A capsular polysaccharide-tetanus toxoid conjugates” Schneerson etal, Infect. Immun. 52: 519 (1986).

The characteristics appropriate to this antigen: high number of monomersof the polysaccharide, nature of the carrier protein, nature of the linkbetween the polysaccharide and the carrier protein, confers particularqualities on it and especially a very good immunogenicity.

In order to retain these qualities in liquid medium in the course oftime, it has now been found that it is possible to use aluminiumcomplexes whose point of zero charge is less than approximately 7.2. Infact, it was found that, surprisingly, when the PRP-T is associated withsuch aluminium complexes, its very good immunogenicity character isretained in the course of time in liquid medium and this takes placewhatever its degree of immobilization on the aluminium complexes.

The point of zero charge of aluminium complexes is the equivalent of theisoelectric point of proteins; it is the pH at which the charge at thesurface of the aluminium complexes is zero. In fact, this point of zerocharge is approached by measurements of zeta potential which can becarried out according to different techniques, the basic method beingelectrophoresis. It is possible to carry out the measurements by meansof an apparatus such as the DELSA 440 of Coulter Electronics, Hialeah,Fla., USA.

The measurement methods and apparatus can be different and the resultsobtained can likewise vary. The aluminium complexes suitable for thepurposes of the invention are those for which this point of zero chargeis less than approximately 7.2, this value of 7.2 being only anapproximate value.

The aluminium complexes suitable for the purposes of the invention arethose which, by nature, have a point of zero charge of less than 7.2 orthose which can be modified in order to lower their point of zerocharge.

Among the aluminium complexes having by nature a point of zero charge ofless than 7.2, it is possible to cite those commonly called aluminiumphosphates in the field of vaccine adjuvants, even if, from a chemicalpoint of view, they have salts other than the aluminium phosphates. Theyare, for example, ADJUFOS® aluminium phosphate supplied by SUPERFOSBIOSECTOR a/s.

They can likewise be aluminium complexes obtained by reaction of sodiumcarbonate in PBS buffer on potassium and aluminium sulphates.

With such complexes, whether the PRP-T is completely or partially boundto the aluminium complexes, its immunogenicity is retained in the courseof time.

Alternatively, it is possible according to the invention to usealuminium complexes which, by nature, have a point of zero charge ofgreater than 7.2 and which are modified to lower this point of charge.

They are especially aluminium complexes known in the field of vaccineadjuvants as being aluminium hydroxides, even if from a chemical pointof view they are not formed exclusively from aluminium hydroxide. Theycan especially be ALHYDROGEL® aluminium hydroxide supplied by SUPERFOSBIOSECTOR a/s, the product used as an adjuvant in the D.T. Coq™ marketedby PASTEUR MERIEUX S & V or even the product used as an adjuvant in theRecombivax® marketed by MERCK.

According to the invention, the modification of these aluminiumcomplexes consists in the addition of anions. The anions added can be ofdifferent nature, on condition that they all have the guarantees ofsafety necessary for use for vaccine purposes. It has been observed thatthe addition of citrate ions or phosphate ions would be particularlywell suited to the purposes of the invention. The phosphate ions canespecially be contributed by a solution containing monopotassiumphosphate, disodium phosphate and sodium chloride.

It is likewise possible to use a combination of different anions, forexample a combination of phosphate ions and carbonate ions.

It is possible for the anions to be added to the suspension of aluminiumcomplexes prior to the addition of the PRP-T, or for the anions to beadded to the PRP-T prior to its contacting with the aluminium complexes.For convenience, it is preferred to suspend the PRP-T in a solutioncontaining the chosen anions before contacting them with the adjuvant.

The quantity of anions added is calculated to lower the point of zerocharge of the aluminium complexes used to a value of less thanapproximately 7.2. This quantity thus varies as a function of the natureof the aluminium complexes used as well as of the quantity of anionspossibly contributed by the buffer substances used. Its determination iswithin the scope of the person skilled in the art.

A vaccine composition stable in the liquid state is thus obtained, thatis to say in which the PRP-T retains its good immunogenic character.

It has additionally been observed that, owing to the addition of anions,the immobilization of the PRP-T on the aluminium complexes was reduced,which may contribute to the maintenance of its integrity and thus of itsimmunogenicity.

Although this is not preferential in the sense of the invention, it islikewise possible to add anions to the aluminium complexes having, bynature, a point of zero charge of already less than approximately 7.2.In this case, it has been observed that the point of zero charge can belowered, and that the immobilization of the PRP-T on the aluminiumcomplexes is likewise reduced.

The term immobilization is understood to mean any form of linkage makingthe PRP-T inaccessible to dosage when, after centrifugation, thesupernatant is collected.

The vaccine compositions according to the invention comprise the vaccineantigen formed by the PRP-T but can likewise comprise other vaccineantigens and especially those intended to protect against diphtheria,tetanus, whooping cough (cellular or acellular), poliomyelitis,hepatitis A, hepatitis B . . . etc. In fact, any vaccine antigencompatible with the PRP-T and the aluminium complexes is capable ofentering into the vaccine composition according to the invention. It isthus possible to have liquid vaccine combinations allowing, in a singleadministration, vaccination against several illnesses. The vaccinecompositions according to the invention are particularly adapted foradministration to young children.

In addition, it has been observed that the use of such a liquid vaccinecomposition during the booster vaccination carried out in infants of 12months who have received injections at 2, 4 and 6 months would allow theproduction of anti-PRP-T antibodies to be increased in a particularlyincreased manner with respect to a booster carried out under the sameconditions with the TETRAct-HIB™ vaccine marketed by PASTEUR MERIEUXSérums et Vaccins.

EXAMPLE 1

A vaccine composition is manufactured starting from the followingconstituents:

Purified tetanus anatoxin (PTA)  1 vaccinating unit Purified diphtheriaanatoxin (PDA)  1 vaccinating unit Pertussis cell mixture 15 OU(opacimetric units) PRP-T (expressed in weight of PRP) 12 μg Merthiolate43.75 μg Aluminium hydroxide (expressed in  0.3 mg Al) such as thatpresent in the D.T. Coq  Phosphates 30 μmol 10 mmolar tris buffercomprising  0.125 ml 8.5% sucrose water for injection qsp  0.5 ml

The phosphate ions are added starting with a solution containingmonopotassium phosphate, disodium phosphate and sodium chloride.

EXAMPLE 2

The immunogenicity of the vaccine composition obtained according toExample 1 was tested in young children in comparison to theimmunogenicity of a vaccine of the previous type formed by the vaccinemarketed under the name TETRAct-HIB™ and which has the same vaccinevalencies but where the lyophilized retained PRP-T valency isreconstituted just before injection with the vaccine compositioncontaining the diphtheria and tetanus anatoxins as well as the Pertussiscellular mixture.

This test was carried out on a group of 262 infants, of which 130received the formulation according to Example 1 and 132 received thecommercial vaccine TETRAct-HIB™. The administration of the vaccines wascarried out at 2, 6 and 12 months intramuscularly.

Before immunization, the GMT titre of anti-PRP antibody was 0.2 μg/ml inthe two groups; it was 1.9 and 1.4 μg after the second injection and 5.9and 5.8 after the 3rd injection respectively for the vaccine accordingto the invention and the vaccine of the prior art.

After the second injection, 98% (with the vaccine according to theinvention) and 93% (with the vaccine according to the prior art) of theinfants had an anti-PRP antibody level of greater than 0.15 μg/ml; thislevel was attained after the 3rd injection in 100% of the infantsreceiving the vaccine according to the invention and in 99% of theinfants receiving the vaccine according to the prior art.

After the 3rd injection, the quantities of antibodies directed againsteach of the vaccine valencies were on average the following:

Prior art vaccine Invention vaccine Diphtheria 1.35 1.56 UI/ml Tetanus5.1 4.9 UI/ml Pertussis agglutin. titre 597 601 GMT PRP 5.8 5.9 μg/ml

After the booster injection carried out at 12 months, the quantities ofantibodies this time were:

Prior art vaccine Invention vaccine Diphtheria 3.2 4.5 UI/ml Tetanus12.0 11.5 UI/ml Pertussis agqlutin. titre 2447 2560 GMT PRP 19.4 32.6μg/ml

These results show that the vaccine combination obtained according tothe invention is stable; in fact the booster injection carried out onchildren 12 months old was carried out with a vaccine which had beenmanufactured 18 months previously; however, the results show that theimmunogenicity of each of the antigens of the combination is retained.In addition, in a surprising manner, a booster effect which is clearlygreater with the vaccine composition according to the invention isobtained, with respect to the booster effect obtained with a vaccine ofthe prior art having the same vaccine valencies.

EXAMPLE 3

Doses of vaccine composition such as described in Example 1 are kept at+4° C. for 18 to 24 months and then used in a clinical trial including104 children.

The titres obtained for each of the vaccine valencies are summarized inthe table below.

Before vaccinatian After vaccination Diphtheria 0.013 0.736 G.M.T.Tetanus 0.181 3.831 G.M.T. PRP G.M.T. μg/ml 0.22 6.40

It can thus be seen that even after storage for a long period at +4° C.,the vaccine composition according to the invention retains itsimmunogenic character, both as far as the PRP-T is concerned as well asthe other vaccine antigens.

EXAMPLE 4

Vaccine compositions each comprising PRP-T at a concentration of 20 μgof PRP/ml are prepared in the presence of aluminium complexes ofdifferent types, and of different points of zero charge (PZC). Thequantity of aluminium complexes is such that the concentration ofaluminium in the composition is 0.6 g/l.

Composition 1: Aluminium complex formed by aluminium hydroxide such asthat used in the D.T. Coq™ vaccine marketed by PMsv. PZC=11.3

Composition 2: Aluminium complex formed by aluminium hydroxide such asthat used in the Recombivax® vaccine marketed by Merck. PZC=7.4

Composition 3: Aluminium complex formed by aluminium phosphate obtainedby mixing sodium chloride and trisodium phosphate. PZC=6.2

Composition 4: Aluminium complex formed by the product called Alum andobtained by reaction of sodium carbonate in PBS buffer on potassium andaluminium sulphates. PZC=5.4

The vaccine compositions are obtained by simple mixture of thesuspensions containing the aluminium complexes and PRP-T.

EXAMPLE 5

The immunogenicity of the different compositions obtained is tested inmice.

In order to verify the immunogenic stability, the compositions obtainedare subjected to conditions of accelerated ageing, i.e. they are kept at37° C. for 2 weeks.

The immunogenicity test is carried out on mice 22-24 g in weight towhich doses of 0.5 ml each containing 2.5 μg of PRP are administeredsubcutaneously. The administrations are carried out on day 0 and on day14. The blood of the mice is taken on day 14 and on day 21 and the levelof antibodies is determined by radioimmunological determination. Thenumber of mice inoculated for each vaccine composition is 8.

The result is considered satisfactory if:

there are at least 75% of the mice on day 21 which have a titre of ≧0.5,

there is a significant difference between the results obtained on day 14and those obtained on day 21.

It is considered that the vaccine composition obtained is stable if theresults obtained after accelerated ageing are satisfactory.

The results obtained for the compositions tested are summarized in thetable below:

PZC Immuno. Test C1 11.3 Not satisfactory C2 7.4 Not satisfactory C3 6.2Satisfactory C4 5.4 Satisfactory

It is thus seen that when the point of zero charge of the aluminiumcomplexes is an acidic pH, the vaccine composition obtained is stable.

EXAMPLE 6

The percentage of PRP-T immobilized on the aluminium complexes isverified for each of the compositions of Example 4.

For this, each of the compositions is centrifuged; the supernatant iscollected in which the quantity of non-immobilized PRP-T is measured byELISA or by RIA.

The difference between the concentration of PRP-T in the startingcomposition and the quantity determined in the supernatant allows thepercentage of immobilized PRP-T to be determined.

The results obtained are stated below:

C1: 100%

C2: 100%

C3: 100%

C4: 70%

EXAMPLE 7

Composition 1 is modified by adding phosphate ions to it in order toobtain a concentration of 50 mmol/l. The immunogenicity test carried outin mice after accelerated ageing of the solution then leads to asatisfactory result.

The determination of the percentage of immobilization of PRP-T onaluminium complexes shows that, under these conditions, only 20% of thePRP-T is immobilized.

EXAMPLE 8

Composition 1 is modified by this time adding citrate ions to it inorder to obtain a concentration of 200 mmol/l.

The immunogenicity test carried out in mice after accelerated ageing ofthe solution then leads to a satisfactory result.

The determination of the percentage of immobilization of PRP-T onaluminium complexes shows that, under these conditions, the PRP-T is nolonger immobilized at all.

EXAMPLE 9

Composition 2 is modified by adding phosphate ions to it in order toobtain a concentration of 20 mmol/l.

The immunogenicity test carried out in mice after accelerated ageing ofthe solution then leads to a satisfactory result. The determination ofthe percentage of immobilization of PRP-T on aluminium complexes showsthat, under these conditions, the PRP-T is no longer immobilized at all.

EXAMPLE 10

Composition 3 is modified by adding phosphate ions to it in differentquantities and the percentage of immobilization of PRP-T on thealuminium complexes is determined.

If the quantity of phosphate ions added is such that the concentrationof phosphates in the composition is 2 mmol/l, the percentage of PRP-Timmobilized is reduced to 10%.

If the quantity of phosphate ions added is such that the concentrationof phosphates in the composition is 4 mmol/l, the PRP-T is no longerimmobilized at all.

The immunogenicity tests carried out in mice after accelerated ageing ofthe solution are satisfactory.

EXAMPLE 11

Composition 4 is modified by adding phosphate ions to it to obtain aconcentration of 60 mmol/l.

The determination of the percentage of PRP-T immobilized on thealuminium complexes shows that, under these conditions, only 30% of thePRP-T is immobilized. The immunogenicity test carried out in mice afteraccelerated ageing of the solution leads to a satisfactory result.

EXAMPLE 12

A vaccine composition is prepared starting from the followingconstituents:

Aluminium hydroxide (expressed in Al)  0.25 mg PRP-T (expressed inweight of PRP) 10 μg PDA  1 vaccinating dose PTA  1 vaccinating dosePhosphates 15 μmoles Polio antigens type I 40 U type II  8 U type III 32U Pertussis anatoxin 25 μg Pertussis F-HA 25 μg 50 mmolar tris buffercomprising 42.5%  0.125 ml sucrose Water for injection qsp  0.5 ml

The immunogenicity tests relative to PRP-T carried out in mice with asolution prepared in this way as well as with a solution stored for 1month at 37° C., a solution stored for 2 months at 25° C., and asolution stored for 6 months at 4° C., all led to satisfactory results,which shows the stability of PRP-T in such an environment.

EXAMPLE 13

A vaccine composition is prepared starting from the followingconstituents:

Aluminium hydroxide (expressed in Al)  0.3 mg PRP-T (expressed in weightof PRP) 10 μg PDA  1 vaccinating dose PTA  1 vaccinating dose Pertussisanatoxin 25 μg F-HA 25 μg Hbs protein (such as presept in the 20 μgGenHevac B PASTEUR ® vaccine) Polio antigens type I 40 U type II  8 Utype III 32 U Phosphates 20 μmol Carbonates  5 μmol 50 mmolar trisbuffer comprising 42.5%  0.125 ml sucrose Water for injection qsp  0.5ml

The stability of the solution prepared in this way is verified bysubmitting it to 2 weeks at 37° C. and by then carrying out animmunogenicity test of PRP-T in mice as described in Example 5.

The results obtained are satisfactory.

An immunogenicity test for the Hbs protein is carried out; this test iscarried out in mice and consists in determining the anti-Hbs antibody byELISA, then in determining the 50% effective dose which may, insofar asthe test is considered satisfactory, be less than 0.970 μg of Hbsprotein.

The results obtained with the solution prepared as indicated above andkept for 2 weeks at 37° C. before being tested were satisfactory.

EXAMPLE 14

A vaccine composition is prepared starting from the followingconstituents:

Aluminium hydroxide (expressed in Al) 0.3 mg PRP-T (expressed in weightof PRP) 10 μg PDA 1 vaccinating dose PTA 1 vaccinating dose Pertussisanatoxin 10 μg F-HA 5 μg Fimbriae 5 μg Pertactin 3 μg Hbs protein (suchas present in the 20 μg GenHevac B PASTEUR^(®)vaccine) Polio antigenstype I 40 U type II 8 U type III 32 U Phosphates 20 μmol Carbonates 10μmol MgCl₂ 5 μmol 50 mmolar tris buffer comprising 42.5% 0.125 mlsucrose Water for injection gsp 0.5 ml

The immunogenicity tests relative to PRP-T carried out as described inExample 5, and those relative to the Hbs protein carried out asdescribed in Example 13, all led to satisfactory results, showing thestability of the vaccine composition according to the invention.

What is claimed is:
 1. A storage-stable, liquid vaccine compositioncomprising capsular polysaccharide of Haemophilus influenzae type b orpolyribosylribitol phosphate having more than 100 monomer repeatingunits coupled to tetanus anatoxin as well as an aluminium-basedadjuvant, wherein the aluminium-based adjuvant has a point of zerocharge of less than approximately 7.2.
 2. The vaccine compositionaccording to claim 1, wherein the aluminium-based adjuvant comprisesaluminium hydroxides to which anions have been added.
 3. The vaccinecomposition according to claim 2, wherein the anions are chosen fromamongst the phosphates and the citrates.
 4. The vaccine compositionaccording to claim 1, wherein the aluminium-based adjuvant comprisesaluminium phosphates.
 5. The vaccine composition according to claim 1,wherein the aluminium-based adjuvant comprises potassium and aluminiumsulphates.
 6. The vaccine composition according to claim 1, wherein saidcomposition additionally comprises one or more of the vaccine valencieschosen from amongst: diphtheria, tetanus, whooping cough, hepatitis Band poliomyelitis.
 7. A process for preparing a storage-stable, liquidvaccine composition comprising capsular polysaccharide of Haemophilusinfluenzae type b or polyribosylribitol phosphate having more than 100monomer repeating units coupled to tetanus anatoxin, said processcomprising obtaining a vaccine composition and adding an adjuvant to thevaccine composition by means of a suspension of aluminium complexeshaving a point of zero charge of less than approximately 7.2.
 8. Theprocess according to claim 7, wherein said adjuvant comprises aluminiumhydroxides to which anions have been added.
 9. The process according toclaim 8, wherein said anions are chosen from amongst the phosphates andthe citrates.
 10. The process according to claim 7, wherein saidaluminium-based adjuvant comprises phosphates.
 11. The process accordingto claim 7, wherein said aluminium-based adjuvant comprises potassiumand aluminium sulfates.